Cells continually engage in clathrin-mediated endocytosis to internalize bits of plasma membrane along with their receptors and signaling molecules. Some two dozen different proteins are though to participate in clathrin-mediated endocytosis, along them actin. Despite intense study in several labs, the role of actin in clathrin-mediated endocytosis remains unclear. This application proposes to test the idea that this actin polymerization provides force, (a) to invaginate coated pits and separate them from the plasma membrane (scission) and/or (b) to propel freshly formed vesicles from the plasma membrane into the cytosol. A specially constructed evanescent field fluorescence microscope will be used to view the plasma membrane and the adjacent 100 nm of cytosol and organelles contained therein. Fibroblasts expressing red fluorescent clathrin will be transfected with GFP-actin and observed in two colors. Single clathrin-coated pits are imaged in living fibroblasts to observe directly the minute movements of coated pits as they invaginate, to detect their separation from the plasma membrane as they become coated vesicles, and to image small assemblies of actin filaments that may form at endocytic sites. Fibroblasts expressing red clathrin will be transfected also with GFP conjugates of other essential proteins in endocytic sites. Fibroblasts expressing red clathrin will be transfected also with GFP conjugates of other essential proteins in endocytosis, in order to determine the sequence with which the proteins are first recruited to endocytic sites and then released. Experiments with inhibitory mutants and microinjected inhibitory peptides will reveal which proteins are required to initiate actin polymerization at endocytic sites, and what happens to a coated pit or vesicle if actin polymerization is prevented. The proposed work will clarify the role of an actin mediated event that is directly related to endocytosis. Clathrin-mediated endocytosis influences multiple cell functions, and actin polymerization is controlled by one of the most extensive regulatory networks used in cells. Defects in either can lead to disease. The proposed work will contribute insights on a fundamental activity performed by all cells.